The invention relates to mutants of tetrameric hemoglobin molecules engineered to possess optimal oxygen carrying characteristics. The molecules are useful as blood substitutes.
Hemoglobin is a tetrameric molecule having a molecular weight of 64,500 Daltons. It is the protein in red blood cells which transports oxygen from the lungs to the tissues. The tetrameric molecule is formed of two pairs of xcex1 and xcex2 subunits. The subunits are held together as a result of ionic, hydrophobic and Van der Waals forces, and not as a result of covalent bonds. When hemoglobin is oxygenated, i.e., combined with oxygen, it readily forms xcex1-xcex2 dimers having a molecular weight of 32,250 Daltons. These dimers are not retained in vivo by the kidneys and are eliminated through the urine. Urine elimination is the result of the dissociation of tetrameric hemoglobins into dimers.
Hemoglobin-based oxygen carriers have been proposed as substitutes for red blood cells, as artificial oxygen-delivery agents for replacing blood transfusions. Acellular products are preferred over cell-based red blood cell substitutes since cell-free products do not require tissue typing, are easier to store than cellular products, have a longer shelf life than cellular products, and can be made virus-free. However, there are problems associated with the use of hemoglobin solutions. Hemoglobin solutions have a short retention time in circulation due to the dissociation of hemoglobin into dimers which rapidly filter through the kidney glomeruli and vasoconstriction due to depletion of nitric oxide in the wall of the vasculature, probably resulting from hemoglobin extravasation.
In human red cells, the interaction of 2,3-diphosphoglycerate (2,3-DPG) with hemoglobin regulates the efficient release of oxygen to the tissues. Inside red blood cells, 2,3-DPG combines with hemoglobin in order to decrease its oxygen affinity to a level compatible with oxygen transport. Solutions of hemoglobin thus have a high oxygen affinity due to the absence of the 2-3-DPG regulation present within red cells.
An acellular product must have a high retention time in the circulation in order to serve as a blood substitute. In order to increase the retention time, it is necessary to impede the dissociation of the tetrameric hemoglobin molecule into dimers. Several types of chemical modifications have been implemented which stabilize the tetrameric form of hemoglobin by introducing an intramolecular cross-link between either the xcex1 or the xcex2-subunits (Chatterjee et al., J. Biol. Chem. 261:9929-9937, 1986; Bucci et al., Biochemistry 5:3418-3425, 1996; Jones et al., J. Biol. Chem. 271:675-680, 1996) High molecular weight polymers have also been obtained by intermolecular chemical crosslinking although these comprise a large distribution of molecules of different molecular weight (Gould et al., Critical Care Clinics 8:293-312, 1992; Gould et al., World J. Surg.20:1200-7, 1996). However, such chemically cross-linked polymers must then be subjected to a series of complex purification steps to remove adjuvants or surfactants associated with the cross-linking process.
In order to maintain the hydric balance, solutions of tetrameric hemoglobin can not be used at a concentration higher than 7%, about half the concentration of hemoglobin in blood. Polymerization of several hemoglobin molecules makes possible the use of higher hemoglobin concentration without exceeding the normal oncotic pressure. By xe2x80x9cpolymerizationxe2x80x9d as used herein with respect to hemoglobin is meant the formation of associations of two or more hemoglobin tetramers. Polymerization also hinders extravasation of tetrameric hemoglobin molecules into the interstitial space apparently decreasing the vasoactivity of these solutions (Doyle et al., J. Biol. Chem. 274:2583-2591, 1999).
Polymerization of tetrameric hemoglobin as a means of impeding dissociation involves the creation of higher molecular weight polymers. However, chemically-induced polymerization results in a large distribution of molecules of different molecular weight. Such heterogenous polymers tend to have varying characteristics across a population, the properties being difficult to control. A homogenous polymer is preferable.
The development of genetic engineering has made available the expression of hemoglobins in microorganisms. Certain mutant forms of hemoglobin have been discussed in the prior art. Hoffman et al. (U.S. Pat. Nos. 5,661,124 and 5,028,588) describe beneficial mutants having a P50 analogous to that of normal hemoglobin. The mutants are characterized by an osmolarity of greater than 303 mmol/L. Bonaventura and Riggs, Science, 158:800-2 (1967) describe the naturally occurring xcex2-chain mutation xe2x80x9cHbPorto Allegrexe2x80x9d in which serine at position 9 is replaced with cysteine.
A recombinant mutant hemoglobin currently under clinical trials has a low oxygen affinity and the tetrameric structure stabilized by using an expression vector containing a single duplicated tandemly fused xcex1-globin gene (Looker et al., Nature 356:258-260, 1992). Another mutant recombinant hemoglobin contains five amino acid substitutions and has an intrinsic low oxygen affinity that is modulated by chloride ions (Fronticelli et al., J. Biol. Chem. 270:30588-30592, 1995). The oxygen affinity values for this mutant are similar to those of whole blood in the presence of physiological chloride ions concentrations (120 mM).
It is an object of the invention to provide a mutant hemoglobin by recombinant DNA techniques having a high oxygen carrying capacity but low oncotic pressure useful as a blood substitute.
It is an object of the invention to provide a mutant hemoglobin which forms polymers of such hemoglobins through disulfide bonds in a controlled fashion, without interference from spurious bonds during refolding and polymerization.
It is a further object of the invention to provide a preparation of stable homogeneous polymers.
It is an object of the invention to provide for the formation of stable hemoglobin polymers without the use of exogenous synthetic chemical crosslinkers.
These and other objects of the invention will be apparent from the following disclosure.
According to the present invention, a modified human xcex2-globin polypeptide is provided having the amino acid sequence of the normal human xcex2-globin modified by (i) the substitution or deletion of Cys at positions 93 and 112, and (ii) the substitution of a Cys for a non-Cys amino acid at one other position. In preferred embodiments, Cys is subsisted for one of the amino acids at the following positions: (a) Ser at position 9; (b) Asn at position 80; or (c) Lys at position 17. Most preferably, Cys is subsisted for Ser at position 9. According to a preferred embodiment, the cysteine residue at position 93 is substituted with an alanine residue, and/or the cysteine residue at position 112 is substituted with a glycine residue. Where all three substitutions are present (Ser9xe2x86x92Cys, Cys93xe2x86x92Ala and Cys112xe2x86x92Gly), the polypeptide has the sequence SEQ ID NO:4. A hemoglobin comprising the aforesaid three-position mutant xcex2-globin and normal xcex1-globin is hereinafter identified as xe2x80x9cHb Priscaxe2x80x9d. Normal hemoglobin is referred to as xe2x80x9cHbAxe2x80x9d. Normal hemoglobin is characterized by xcex1- and xcex2-globin polypeptides having the native, i.e., non-mutant or wild type, amino acid sequences.
In another aspect, the invention is directed to nucleic acid encoding the aforesaid mutant xcex2-globin polypeptide. In preferred embodiments, the nucleic acid encodes the polypeptide of SEQ ID NO:4. In one such embodiment, the nucleic acid has the nucleotide sequence SEQ ID NO:2.
In another aspect, the invention is directed to vectors comprising a promoter operably linked to a nucleic acid sequence encoding the mutant xcex2-globin polypeptide, capable of directing the expression of a mutant human xcex2-globin polypeptide. The invention is further directed to host cells transformed with such vectors.
A method for producing a human xcex2-globin mutant polypeptide is provided comprising a culture of the aforesaid transformed host cells under conditions conducive to the expression of said polypeptide by said host cells.
The invention is also directed to a modified human hemoglobin in which at least one of the xcex2-globins, preferably both xcex2-globins, is a modified xcex2-globin as described above. By xe2x80x9chemoglobinxe2x80x9d is meant the functional tetrameric molecule comprising two xcex1-globin polypeptides, two xcex2-globin polypeptides and heme. In one embodiment, the modified human hemoglobin is further characterized by the presence of an xcex1-globin mutant polypeptide having the amino acid sequence of normal human xcex1-globin modified by the substitution or deletion of Cys at position 104. A preferred mutation is Cys104xe2x86x92Ser (SEQ ID NO:6). The xcex1-globin may be further modified by either the substitution of Cys for Ala at position 71, or the substitution of Cys for Ala at position 53. Both xcex1-globin polypeptides of the tetrameric hemoglobin are preferably mutant.
According to one embodiment, the modified hemoglobin has all of the native Cys residues substituted by non-Cys residues, and one non-native Cys is introduced into either the xcex1-globin or xcex2-globin polypeptide, by substitution. Thus, a modified human hemoglobin comprises a mutant human xcex1-globin polypeptide comprising the amino acid sequence of normal human xcex1-globin modified by the substitution of Cys at position 104 by a non-Cys amino acid; and a mutant human xcex2-globin polypeptide comprising the amino acid sequence of normal human xcex2-globin modified by the substitution of Cys at positions 93 and 112 by non-Cys amino acids. The modified hemoglobin is further characterized by the substitution of Cys for the native sequence amino acid at one of the following positions: xcex2-globin position 9; xcex2-globin position 17; xcex2-globin position 80; xcex1-globin position 71; or xcex1-globin position 53. Thus, since each hemoglobin contains two xcex1 and two xcex2 chains, each molecule of modified human hemoglobin contains two non-native Cys residues.
The invention is further directed to a polymeric hemoglobin comprising the aforesaid modified human hemoglobins. The polymer may contain, for example, from 2 to 200 hemoglobin molecules, more preferably from 2 to 40, most preferably from 2-20. In one embodiment, the modified tetrameric hemoglobins of the polymeric hemoglobin are covalently bonded to each other by one or more intermolecular disulfide bridges formed by cysteine amino acid residues. In one embodiment, the modified hemoglobin of the polymer HB Prisca, and the polymer comprises from two to seven of the modified hemoglobins
The invention is also directed to an artificial blood substitute comprising the aforesaid polymeric hemoglobin, preferably in combination with a pharmaceutically acceptable carrier. A method of supplementing the oxygen-carrying capacity of a patient""s blood comprises administering to the patient an effective amount of the aforesaid blood substitute.